Patients with acute lung injury develop hypoxemia and gas exchange impairment which results in significant morbidity and mortality. It has been reported that hypoxia may impair the lung's ability to clear edema by inhibiting Na+ channels and the Na,K-ATPase in the alveolar epithelium. The focus of this application is to determine whether severe hypoxia of 1.5% or 3% (about 10 or 20 mm Hg) for 1 to 4 hours inhibits Na,K-ATPase by causing endocytosis of the Na+ pump into intracellular compartments via specific pathways activated by reactive oxygen species (ROS) and protein kinase C (PKC) signaling molecules. We will determine whether after exposure to hypoxia, reoxygenation of the alveolar epithelial cells (AEC) results in the recruitment of previously endocytosed Na+ pumps back into the cell basolateral membranes (BLM). We will study whether activation of the protein kinase A pathway by terbutaline and forskolin reverses the effects of hypoxia on AEC Na+ pumps and whether this increases lung liquid clearance. We will also determine whether in AEC exposed to prolonged hypoxia (>12 hours) the Na,K-ATPase proteins undergo ubiquitination and degradation via the proteasomal or lysosomal pathways. As such, we will study the effects of hypoxia on the alveolar epithelium by focusing on the mechanisms of Na,K-ATPase regulation in four interrelated aims: in Specific Aim # 1 we propose to determine the role of mitochondrial ROS generated during hypoxia on Na,K-ATPase function and protein endocytosis; in Specific Aim # 2 we will study whether the alveolar epithelial Na,K-ATPase is phosphorylated during hypoxia by PKC triggering the endocytosis of the Na+ pump; in Specific Aim # 3 we will determine whether hypoxia-mediated inhibition of the Na,K-ATPase function and endocytosis of the Na+ pump are reversible upon reoxygenation and whether activation of the PKA pathway by terbutaline and forskolin prevents or reverses the effects of hypoxia; in Specific Aim # 4 we propose to determine whether prolonged exposure of AEC to hypoxia leads to ubiquitination and degradation of the Na,K-ATPase proteins via the proteosomal/lysosomal pathways. Experiments have been conducted for each of the specific aims and the preliminary results support the feasibility of this proposal. Completion of the proposed studies will provide novel information on the effects of hypoxia on the alveolar epithelium, specifically as it pertains to mechanisms of inhibition and degradation of the Na+ pump as well as pathways of reversal of Na,K-ATPase inhibition, which may be of importance for the design of novel approaches to increase edema clearance in patients with pulmonary edema.